We have discovered a 148 aa protein named calcitonin gene-related peptide (CGRP)-receptor component protein (RCP) that is required for G protein-coupled signal transduction at receptors for the neuropeptide CGRP. During the previous project period we determined that RCP works in conjunction with two other proteins to constitute a functional CGRP receptor: calcitonin receptor-like receptor (CRLR) and receptor activity modifying protein (RAMP1). CRLR has the stereotypical 7-transmembrane topology of a G protein-coupled receptor, and requires RAMP1 for trafficking to the cell surface and for ligand specificity, and requires RCP for coupling to the cellular signal transduction pathway. CRLR did not function in antisense cell lines that inhibited RCP expression, and CRLR co-immunoprecipitated with RCP suggesting that the two proteins were together in complex. The specific hypotheses to be tested in this proposal are that a functional CGRP receptor is composed of at least three proteins: CRLR, RCP, and RAMP1, and that the accessory proteins RCP and RAMP1 can modulate CRLR function. The specific aims of this proposal are to: 1) determine the sites of interaction between RCP and CRLR required for a functional CGRP receptor, using yeast two-hybrid assay and cell culture, 2) determine if overexpression of RAMP1 or RCP can increase CGRP receptor function in cell culture, 3) determine if loss of RCP by homologous recombination has increased inhibitory effects on CGRP receptor function in cell culture, 4) identify the cellular environment that regulates the CGRP receptor complex, focusing on lipid rafts and their effect on receptor function and receptor distribution in the membrane. [unreadable] [unreadable] Recent studies have found that in hypertension the vasculature is hyper-responsive to CGRP, suggesting regulation of CGRP receptor function. Thus, the role of RCP in regulating CGRP receptor function is important both for fundamental studies on G protein-mediated signal transduction in neuroendocrine cells, but also for future therapeutic strategies for vascular disorders.